high-power pulsed infrared laser  (Thermo Fisher)

Journal: bioRxiv

Article Title: The vestibulospinal nucleus is a locus of balance development

doi: 10.1101/2023.12.06.570482

Figure Lengend Snippet: (A) Tg(nefma::EGFP) labels fewer (14.8 ± 0.7, n=20 hemispheres, 10 fish) neurons than optically-backfilled Tg(α-tubulin:C3PA-GFP) fish (24 ± 0.6, n=25 hemispheres, 13 fish, unpaired t-test p=8.3×10 −12 ). (B)Dorsoventral distribution of vestibulospinal cells in both lines. Tg(nefma::EGFP) labels fewer cells in the dorsal vestibulospinal nucleus compared to photofills in Tg(α-tubulin:C3PA-GFP) . (C)Representative maximum intensity projection of spinal projecting neurons in the hindbrain of a Tg(α-tubulin:C3PA-GFP) fish following optical backfill before (top) and after (bottom) two-photon mediated photoablation. (D)Average probability distributions (± S.D) of inter-bout pitch angle for sibling controls (black, N=17 fish) and vestibulospinal lesioned fish (orange, N=17 fish) show no change in average posture (dashed vertical lines) but greater variability (solid horizontal lines ± 1 S.D.). (E)Standard deviation of pitch is higher in vestibulospinal lesioned fish (20.2± 1.2°) compared to sibling controls (13.5 ±1.7°, unpaired t-test p=5.5×10 −9 ). (F)Bout rate as a function of deviation from preferred posture for lesions (orange) and control siblings (black). Solid lines represent raw data, dashed lines represent parabolic fits to raw data ± S.D. of the fit estimates. (G)Pitch sensitivity (parabolic fit) is decreased in vestibulospinal lesioned fish (0.18 ± 0.05, unpaired t-test p=9.9×10 −5 ) compared to sibling controls (0.42 ± 0.15).

Article Snippet: Photoablations were performed on an upright microscope (ThorLabs) using a 80 MHz Ti:Sapphire oscillator-based laser at 920 nm for cell visualization (SpectraPhysics MaiTai HP) and a second, high-power pulsed infrared laser for two-photon mediated photoablation (SpectraPhysics Spirit 8W) at 1040 nm (200 kHz repetition rate, 500 pulse picker, 400 fs pulse duration, 4 pulses per neuron over 10 ms) at 25-75 nJ per pulse, depending on tissue depth.

Techniques: Standard Deviation